The present invention relates to DNA molecules, particularly from Spinacia oleracea, containing the coding region of a 2-oxoglutarate/malate transporter and the introduction of which into a plant genome modifies the production and transport of carbon skeletons for nitrogen fixation and the transport of the assimilated nitrogen in transgenic plants. The invention furthermore relates to plasmids, yeasts and bacteria containing said DNA molecules, as well as to transgenic plants in which modifications of the activity of the 2-oxoglutarate/malate transporter and thus modifications of the nitrogen and carbon metabolism are brought about by introduction of said DNA molecules. The invention furthermore relates to transgenic plants the photo-respiratory capability of which is influenced by the modification of the activity of the 2-oxoglutarate/malate transporter. The invention also relates to the use of the DNA molecules described which code for a 2-oxoglutarate/malate translocator for the identification of related translocators from Spinacia oleracea and other plants by low-stringency hybridization or by PCR techniques, as well as the use of the 2-oxoglutarate/malate translocator as target for herbicides.
Only plants, bacteria and yeasts are capable of converting on a large scale inorganic nitrogen (nitrate nitrogen) into organically fixed nitrogen (usually in the form of amino acids) by reductively aminating organic carbon compounds. The remainder of the animated world, particularly useful animals and humans, is dependent on plants as primary suppliers of organic nitrogen compounds. Assimilation of inorganic nitrogen in plants by fixation to organic carbon depends on the availability of nitrogen, carbon skeletons and energy.
The nitrogen supply of the plant can be influenced by fertilizers. The energy for nitrogen assimilation is derived from the light reaction of photosynthesis or in roots or other non-green tissues from dissimilation and under normal field conditions is no limiting factor.
2-oxoglutarate (.alpha.-ketoglutarate) according to today's knowledge is the primary acceptor of reduced nitrogen in the glutamine synthase/glutamine 2-oxoglutarate aminotransferase (GOGAT) reaction. In this reaction nitrogen (ammonium nitrogen) reduced by glutamine synthase is first transferred onto glutamate under energy consumption. Glutamine is formed. In a sequential reaction the glutamate oxoglutarate aminotransferase (GOGAT; glutamate synthase) catalyzes the transfer of an amino group of glutamine onto 2-oxoglutarate (transamination) while consuming reduction equivalents. Two glutamate molecules are formed.
The entire reaction sequence of the glutamine synthase/GOGAT reaction is localized in the stroma of the plant plastids. These organelles are surrounded by two lipid bilayer membranes, with the exterior having molecular sieve character and being permeable to compounds up to a size of about 10 kD (Flugge and Benz, 1984, FEBS Lett. 169:85-89). The inner membrane is permeable to some smaller compounds such as water, carbon dioxide, oxygen and nitrite, however, not to larger charged molecules such as 2-oxoglutarate.
The key compound of the glutamine synthase/GOGAT reaction, the 2-oxoglutarate, must be moved from the cytosol of the plant cell by a specific translocator across the inner membrane of the chloroplast envelope into the stroma of the plastid. Transport of 2-oxoglutarate into the plastids takes place in exchange with malate from the plastids via the 2-oxoglutarate/malate translocator. The malate exported in this process into the cytosol is transported back by a second translocator, the dicarboxylate translocator, which is related with the 2-oxoglutarate/malate translocator in its substrate specificity, in exchange with glutamate. As a result, 2-oxoglutarate is imported into the chloroplast and the end product of the glutamine synthase/GOGAT reaction, glutamate, is exported without a net transport of malate which circulates via both translocator systems ("double translocator", Woo et al., 1987, Plant Physiol. 84:624-632; Flugge et al., 1988, Planta 174:534-541). Glutamate is the plant's preferred amino group donor in a series of transamination reactions, for example in the biosynthesis of the amino acids alanine or phenylalanine, etc. Furthermore, glutamate is an important transport form for organically bound nitrogen within the plant. Most nitrogen-containing compounds in the plant such as amino acids, nucleic acids or alkaloids require glutamate as primary amino acid donor for their biosynthesis pathway.
The 2-oxoglutarate required for nitrogen assimilation is essentially synthesized by conversion of citrate in the cytoplasm of the cells.
More recent publications (Riesmeier et al., 1993, Proc. Natl. Acad. Sci. USA 90:6160-6164; Heineke et al., 1994,Planta 193:174-180) show that the effectivity of the photosynthetic carbon reaction is inter alia substantially limited by the export of the reduced, organically bound carbon (triose phosphate) formed which is catalyzed by a translocator localized in the inner membrane of the chloroplast envelope. This translocator protein thus is a "bottle-neck" in the carbon metabolism. The plastid 2-oxoglutarate/malate translocator plays a similar role in the nitrogen metabolism.
The plastid 2-oxoglutarate/malate translocator thus plays a key role in the nitrogen metabolism of plants since it is responsible for supplying sufficient amounts of the substrate for nitrogen assimilation by the glutamine synthase/GOGAT reaction. By manipulating the activity of this translocator it would therefore presumably be possible to influence the effectivity of nitrogen assimilation in plants.
Since the majority of humans on earth has to depend on a vegetarian diet, resulting in a continuous inadequate provision with proteins in these social strata, there is an urgent demand for plants having an increased content in organic nitrogen compounds, particularly proteins and amino acids. In the industrialized countries animal and fish meal is increasingly being added to animal feeding stuff to improve the provision of breeding animals with proteins. Forage plants having a higher protein content would surely be the better alternative in particular considering the problems, such as BSE, arising from the feeding of animal meals.
It would be possible to influence the activity of the plastid 2-oxoglutarate/malate translocator via genetic engineering techniques if DNA sequences coding for such a translator were available. So far, this has not been the case. The provision of DNA sequences coding for a 2-oxoglutarate/malate translocator would furthermore allow identification of substances which specifically inhibit said translocator and which can hence be used as herbicides.
Presently, sequences of translocator proteins of the substrate specificity described above are known only from the mitochondria of bovine hearts and from human mitochondria (Runswick et al., 1990, Biochemistry 29:11033-11040;Iacobazzi et al., 1992, DNA Seq. 3(2):79-88). These transporters play an essential role in the mitochondrial dicarboxylate metabolism (inter alia malate/aspartate shuttle, oxoglutarate/isocitrate shuttle) and pertain to the family of mitochondrial metabolite transporters which are closely interrelated. For example, the mitochondrial carriers (phosphate/OH.sup.-, ADP/ATP, oxoglutarate/malate, etc.) are characterized by sequence relationship and by the presence of internal repeats. Furthermore, it could be shown for the most mitochondrial carriers as for the 2-oxoglutarate carrier that they are incorporated into the mitochondrial membrane without a presequence (targeting sequence) directing them to the organelles (Runswick et al., 1990, Biochemistry 29:11033-11040). It can thus be presumed that the targeting information is contained in the mature carrier protein. An over-expression of a mitochondrial dicarboxylate transporter in plants would thus not result in an increase in the oxoglutarate transport across the plastid envelope membrane but only in an increase in the mitochondrial dicarboxylate transport, which is an undesired effect.
In contrast to the above, the proteins of the inner envelope membrane of the plastid require a presequence specifically directing them to the plastids (overview articles: Keegstra et al., 1989, Annu. Rev. Plant. Physiol. Plant Mol. Biol. 40:471-501; Lubben et al., 1988, Photosynth. Res. 17:173-194;Flugge , 1990, J. Cell Sci. 96:351-354). In addition to the presequence needed for "plastid targeting", there is further information in the mature part (the part of the protein remaining after cleavage of the presequence by a specific protease) of the plastid envelope membrane proteins which prevents transport of the envelope membrane proteins across the envelope membrane into the plastid stroma or the thylakoid membrane (own, unpublished observations; Li et al., 1992, J. Biol. Chem. 267:18999-19004). So far it has not been possible to exactly localize this information in the mature part of the proteins of the inner envelope membrane known so far (37 kD protein: Dreses-Werringloer et al., 1991, Eur. J. Biochem. 195:361-368; triose-phosphate/phosphate translocator: Flugge et al., 1989, EMBO J. 8:39-46; Willey et al., 1991, Planta 183:451-461; Fischer et al., 1994, Plant Jour. 5(2):215-226; Ca.sup.2+ ATPase: Huang et al., 1993, Proc. Natl. Acad. Sci. USA 90:10066-10070). Our own investigations conducted with a mitochondrial carrier, the ADP/ATP transporter, showed that this protein cannot or only very ineffectively be directed to the chloroplasts and there be incorporated in the envelope membrane. Even a hybrid protein, consisting of a chloroplastid presequence (containing the information for chloroplast targeting) and said mitochondrial carrier showed only a slight increase in incorporation into the chloroplast envelope membrane as compared to the authentic protein (unpublished observations). Since the protein/lipid interaction is important for a correct insertion and the chloroplastid envelope membrane fundamentally differs in its lipid composition from that of the mitochondria (Joyard et al., 1991, Eur. J. Biochem. 199:489-509), it is highly improbable that an insertion of a mitochondrial protein, albeit a minor one, will be functional, i.e., that a transporter from other organelles can be incorporated at all into the chloroplast envelope membrane in a conformation and orientation corresponding to its function.
Thus, according to the present state of the art, it is not possible to functionally integrate, e.g., mitochondrial or procaryotic dicarboxylate transporters into the inner envelope membrane of the chloroplasts by using known plastid targeting sequences. With the present state of the art it is more promising to achieve the DNA sequence required for the construction of the plants described above by cloning the authentic plastid 2-oxoglutarate/malate translocator.
This cloning, however, cannot be performed by low-stringency screening of a plant cDNA library with a probe derived from the mitrochondrial dicarboxylate transporters. According to what is known today, the chloroplastid translocators have a primary sequence that is completely different from all translocators from other systems (bacteria, mitochondria)--even if the protein has a comparable function. Therefore, the route of biochemical characterization, purification and isolation of the 2-oxoglutarate/malate translocator had to be taken, which is extremely difficult in membrane proteins. Identification of the translocator protein as component of the inner envelope membrane of the chloroplast having an apparent molecular weight of 45,000 Dalton was now possible (Menzlaff and Flugge, 1993, Biochim. Biophys. Acta 1147:13-18). However, the purification procedure described is not suitable to produce sufficient amounts of protein for protein sequencing, since it turned out that the N terminus of the protein was blocked and thus was not available for N-terminal protein sequencing by automated Edman degradation. Therefore, it has not been possible so far to isolate, starting from the amino acid sequence, DNA molecules coding for a plastid 2-oxoglutarate/malate translocator.
The problem underlying the present invention therefore is to provide processes and DNA molecules the use of which makes it possible to modify plants such that they are capable of synthesizing an increased amount of organic nitrogen compounds. Specifically, the problem underlying the invention is to provide DNA molecules coding for a plastid 2-oxoglutarate/malate translocator.
The problem is solved by the provision of the embodiments characterized in the patent claims.